Communication between cells in a tissue takes place in two different ways: release of molecules such as neurotransmitters, hormones or calcium ions;formation of intercellular channels in the plasma membrane enveloping the cell that connect directly the cytoplasm of two adjacent cells. Multicellular organisms have evolved to use different types of intercellular channels, called gap junctions. In vertebrates, a gap junction channel is formed by the apposition of two hexamers of gap junction proteins, one from each adjacent cell. Gap junctions are found in essentially all tissues suggesting an enormous diversity of function. These intercellular channels are critical for integrating and regulating basic cell processes such as metabolic cooperation, ionic transmission, differentiation and hormonal regulation. Breakdown in gap junctional communication can lead to developmental defects, abnormal cell proliferation such as cancer and the failure of tissues to function in their normal capability. Examples of the latter come from the study of patients with hereditary gap junction diseases such as the dysfunction of peripherial nerves (X-linked Charcot-Marie-Tooth Disease) and certain forms of malformation of the heart, deafness, cataracts and skin diseases. We focus on the structure and function of Connexin26 (Cx26), the second smallest of the gap junction protein family. Mutations in the DNA of Cx26 account for about one half of cases of prelingual inherited deafness in Caucasian populations. We have isolated preparations of Cx26 gap junctions and hemichannels in sufficient quantities for biochemical and structural studies by electron microscopy (EM) and atomic force microscopy (AFM). In Specific Aim 1, we will determine the structure of the Cx26 channel beyond 8 E using state of the art cryo-electron crystallography. In Specific Aim 2, we continue our lower resolution analyses of mutant Cx26 channels and hemichannels in order to determine differences in pore structure and connexon stability. In Specific Aim 3, a 3D EM analysis of several isoforms will determine if differences in pore size and shape are related to their physiological properties. Finally, Specific Aim 4 is focused on both imaging under in vitro physiological conditions and on protein unfolding of gap junctions using AFM and these results will be correlated with our EM structures. Each of these aims is intended to complement the others leading to improved structural and physiological models of Cx26 channels germane to the entire connexin family. Gap junction channels directly regulate cell-cell activities by passing metabolites, ions and signaling molecules. This project investigates the structure-function basis of intercellular communication between cells in tissues and organs by focusing on how gap junction intercellular membrane channels are put together from their constituent proteins, the connexins, and how specific parts of the connexin are involved in gating, docking or regulation of the channel.